ABSTRACT Members of ATP binding cassette (ABC) transporter family play crucial roles in prokaryotic and eukaryotic cellular processes. They harness the energy from ATP hydrolysis to transport a wide range of cargo molecules across extra- and intracellular membranes, including metabolic products, lipids and sterols, and xenobiotics. In humans, ABC transporters are involved in chemotherapy resistance, pathology of cystic fibrosis and of other inherited human diseases. This family is also responsible for multi-drug resistance in both bacterial and human. Thus, it is fundamental in understanding the structure and mechanism of ABC transporters. It will be insightful for developing therapeutics to target this family. The goal of this application is to map the functional cycles of eukaryotic and prokaryotic ABC transporters, for the purpose of dissecting the underlying mechanism that couples the ATP hydrolysis with transporting cargo molecules across membrane. A key step is to determine high-resolution structures of the same ABC transporter at different functional stage of its cargo pumping cycle. ABC transporter is a dimeric integral membrane protein formed by either two identical or different monomers. Structure determination of integral membrane proteins and their complexes has always been a tremendous challenge in X-ray crystallography. Crystallization of the same ABC transporter protein at different functional stages is particularly challenging. In this project, we will use an alternative structural biology approach, single particle electron cryo-microscopy (cryoEM) to derive high-resolution structural information of ABC transporter in various conformational states corresponding to intermediates of the transporter cycle. This technique does not require crystal formation and uses minute amounts of protein. We have already achieved 8 resolution of one ABC transporter making use of antibody fragments (Fabs) as fiducial markers. We will use the state-of-the-art high-resolution cryo-EM to: (1) determine structures of two ABC transporters, one prokaryotic and the other eukaryotic, locked into different conformational states using nucleotide analogs, cross-linking, and conformationally-specific Fabs; (2) determine near-atomic resolution structures of these ABC transporters by single particle cryoEM without crystallization, and (3) determine the structures of these two ABC transporter in the membrane environment. Successful completion of these aims will advance our knowledge of the ABC transporters, their pathophysiology and ultimately development of therapeutics for this important class of drug targets.